Metabolic homeostasis plays a central role in all aspects of life, allowing animals to balance their dietary intake with the energy needs required for day-to-day survival. Conversely, mis-regulation of metabolism can lead to obesity and type 2 diabetes, which are critical risk factors for human disease, including cardiovascular disorders and cancer. Our research exploits the speed and power of Drosophila genetics to identify and characterize key aspects of metabolic control with the goal of identifying new levels of regulation that are conserved through evolution, from flies to humans. The molecular context for our studies are nuclear receptors (NRs), which are ligand-regulated transcription factors that play a central role in maintaining metabolic homeostasis. Drosophila has 18 NR genes, significantly fewer than the 48 genes found in humans, spanning all vertebrate NR subfamilies and encoding homologs of key human receptors, including HNF4 (dHNF4), LXR (DHR96), NR4A receptors (DHR38), TR2/TR4 receptors (DHR78), and ERR (dERR). This provides a simplified context for functional studies of NR signaling pathways, allowing us to use Drosophila to define the ancestral roles of evolutionarily-conserved NR subclasses in the absence of genetic redundancy. Over the past four years of NIDDK support our studies of dHNF4, DHR96, DHR78, and dERR, have demonstrated that their fundamental activities have been conserved through evolution, and have revealed new insights into the regulation and function of their mammalian counterparts. In this renewal application, we propose two specific aims that continue our studies of Drosophila NRs with a focus on lipid metabolic pathways. These aims build off recent studies in our lab that identified new activities for the Drosophila members of the ERR and TR2/TR4 NR subfamilies. In aim 1, we will test the hypothesis that dERR supports lipid uptake, transport, and/or storage in adults, analogous to the role of ERR? in the intestine and adipose tissue. In aim 2, we will test the hypothesis that DHR78 plays a central role in lipid metabolism and mitochondrial activity that supports female fertility and suppresses neurodegeneration. By combining metabolite measurements, metabolomic profiling, RNA-seq, ChIP-seq, tissue-specific rescue experiments, RNAi studies, functional characterization of select target genes, and ligand regulation of the receptor, we will define the mechanisms of NR signaling at a level of resolution that is difficult to achieve in more complex organisms. These advances, in turn, will improve our design of mouse models for future functional studies, harness the power of model organism genetics to understand the factors that contribute to diabetes and obesity, and facilitate the development of therapeutic approaches to treat metabolic dysfunction.